Introduction
Although some infections are unique enough to be identified clinically, usually microbiologic laboratory methods are needed to identify the etiologic agent and diagnose microbial infection (Washington, J.A., 1996). Although we have made significant progress in our ability to diagnose and treat infectious diseases, they still remain a strong challenge to human survival, for example the disease Tuberculosis caused by a microbial infection with Mycobacterium tuberculosis accounted for one third of the worlds bacterial infections in 2010 infecting a total of 8.8 million people worldwide (Dheda et al., 2010).
Diagnosis is important not only for prescribing effective drugs but for preventing the evolution of resistant microorganisms (Mori and Notomi, 2009). Traditionally, the microbiology laboratory identified etiologic agents of infectious disease by the direct examination and culture of clinical specimens. Methods of identifying and differentiating microorganisms responsible for microbial infection mainly relied on microbial morphology, staining properties of the organism and its growth variables. However, a major restriction is that >99% of the microorganisms observed through a microscope are not cultivable by these direct techniques (Rastogi and Sani, 2011).
Other disadvantages of these traditional methods include their lack of reproducibility and the difficulties they produce in typing as they are usually not sensitive enough for strain differentiation. These techniques are also restricted by the number of organisms present and the skill of the microbiologist in recognising different pathogens (Tang et al., 1997). Over the past few decades, the development and introduction of molecular-based techniques into the labora...
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...nd Notomi, T. (2009) 'Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases.', J Infect Chemother, 15(2), pp. 62-69.
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Rastogi, G. and Sani, R.K. (2011) 'Chapter 2 :Molecular Techniques to Assess Microbial Community Structure, Function, and Dynamics in the Environment', in Ahmad, I., Ahmad, F. and Pichtel, J. (ed.)Microbes and Microbial Technology. California, USA: Springer Science, pp. 29-57.
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Washington, J.A. (1996) 'Chapter 10 :Principles of Diagnosis', in Samuel Baron (ed.) Medical Microbiology 4th Edn. Galveston, Texas: University of Texas Medical Branch.
The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species.
...uciana L. "Challenges to the Laboratory Diagnosis of Yersinia Pestis in the Clinical Laboratory." Lab Diagnosis of Y. Pestis. UPMC Center for Health Security, 29 Dec. 2005. Web. 07 Apr. 2014.
Trabelsi, H., Dendana, F., Sellami, A., Sellami, H., Cheikhrouhou, F., Neji, S., … Ayadi, A. (2012). Pathogenic
Bacteria play a large role in our health, the environment, and most aspects of life. They can be used in beneficial ways, such as decomposing wastes, enhancing fertilizer for crops, and breaking down of substances that our bodies cannot. However, many bacteria can also be very harmful by causing disease. Understanding how to identify bacteria has numerous applications and is incredibly important for anyone planning to enter the medical field or begin a career in research. Having the background knowledge of identifying an unknown bacteria may one day aid healthcare professionals diagnose their patient with a particular bacterial infection or help researchers determine various clinical, agricultural, and numerous other uses for bacteria.
Results from Gram staining procedures were provided a week later; hence, a dichotomous (a flowchart that is used to identify bacteria based on its taxonomic classification) was constructed based on a total of 10 possible unknown bacterium. The bacteria were then separated based on its Gram reaction; leading to two subcategories, including Gram positive and Gram negative. Both subcategories were then divided into two separate categories based on bacteria morphology, including rod and cocci shaped. A series of metabolic test were then selected strategically based on metabolic results provided by Dr. McLaughlin (See attached chart for possible results). The metabolic test for Gram positive rod
Morse, Stephen A., et al. "Detecting biothreat agents: the laboratory response network." ASM News-American Society for Microbiology 69.9 (2003): 433-437.
An unknown broth culture labeled # 17 was supplied by the lab instructor to identify the bacteria. We prepared a SOP by incorporating laboratory techniques that were considered as determinative tests in the dichotomous keys (appendix B and appendix C). The first two steps we performed to form the basis of identification were Gram’s staining and plate streaking of the sample. The supplied broth culture was streaked out simultaneously on one of each Trypticase Soy Agar (TSA), Mannitol Salt Agar (MSA) and MacConkey Agar (MAC) plate. The streaking was performed using the quadrant streak method described in the lab manual (Leboffe & Pierce, 2010). After observing the colony morphology and growth pattern, we further
Observing under 1000X magnification, the gram stain depicts purple bacilli, as seen in Figure 1. Figure 2 depicts growth of a light brown, opaque bacteria on a Phenylethyl Alcohol Agar (PEA) plate. As seen in Figure 3, the blood agar plate shows brown growth surrounded by a very thin clear area. Figure 4 shows an endospore stain of the gram-positive unknown. It depicts green endospores surrounded by pink bacilli. Figure 5 depicts a slant of gram-positive isolate, which appears as an opaque, white film. As seen in Figure 6, the catalase test resulted in vigorous bubbling. The lactose test resulted in a color change from pink to yellow, as seen in Figure 7.
Mycobacterium tuberculosis is a nonmotile, acid-fast, obligate aerobe. The bacilli are 2-4 um in length and have a very slow generation time of between 15 and 20 hours. The cell wall of the mycobacterium is unique in that it is composed mainly of acidic waxes, specifically mycolic acids. M. tuberculosis is unusually resistant to drying and chemicals, contributing to the ease with which it is transmitted.
Polymerase chain reaction (PCR) is a technique used “to amplify the number of copies a specific region of DNA (Brown)”, in order to produce enough DNA to be adequately tested. This technique can be used to identify with a very high-probability, disease-causing viruses, bacteria, a deceased person, a criminal suspect, and also in the event of an outbreak, “Real-Time PCR can effectively monitor the success of clean-up efforts (RAL,Inc)”.
Rouphael, N.G. & Stephens, S.S. (2012). Neisseria meningitidis: Biology, Microbiology, and Epidemiology. Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology. 799: 1-20. Retrieved from: http://download.springer.com/static/pdf/954/chp%253A10.1007%252F978-1-61779-346-2_1.pdf?auth66=1385454607_76421ed179b1332c8755d5ca9118b502&ext=.pdf
stains on sputum’s and body fluids, and have completed a few AFB cultures. Apart from
Greenwood, D., Slack, R., Peutherer, J. and Barer, M. (2007) Medical Microbiology, the United States: Elsevier.